Most prevalent non-infectious diseases affecting people in the United States are associated with cellular damage and organelle dysfunction. Yet the mechanism by which these diseases occur and why their onset dramatically increase as humans age is unclear. The budding yeast Saccharomyces cerevisiae is an important model system for studying organelle biogenesis and function. Furthermore, the nature of its asymmetric cell divisions indicates that the vitality of a mother cell declines with each cell division, and its limited replicative life span has been used as a model to study and identify conserved genetic and environmental processes that extend life span in metazoans. However, its use in studying the process of decline has been limited, in large part due to the difficulty in isolating mother cells that have gone through an increasing number of cell divisions. We have recently overcome this limitation by development of the Mother Enrichment Program (MEP), and can now isolate and examine large populations of synchronously aged cells. We have applied our novel technology in combination with the full set of resources available in S. cerevisiae to study molecular mechanisms of spontaneous organelle decline. We discovered the earliest changes occurred at middle-age and coincidentally: mitochondrial fragmentation and loss of mitochondrial membrane fusion, loss of vacuolar acidity and accumulation of a subclass of ER-Golgi vesicles. We will determine whether the mitochondria, vacuole and ER-Golgi are causally linked in their decline, and if so, we will define the mechanism(s) that links them. This will lay the groundwork for our goal of identifying specific molecules that become defective with repeated cell divisions, how they become defective and the impact of the defect(s). Because these organelles are conserved and implicated in human diseases, our unique ability to conduct these studies may very well provide new insights about age-associated decline in humans. We also discovered dramatic alterations in two critical proteins of mitochondrial membrane fusion (Fzo1 & Ugo1). These changes offer a likely explanation for the loss of mitochondrial fusion we observe as mother cells divide. We propose to identify the chemical nature of, and processes responsible for, the alterations in these proteins, employing a combination of biochemical and genetic approaches. Similar mitochondrial fragmentation and fusion defects have been linked to neuropathies, diabetes and muscle deterioration. Thus our findings may provide a paradigm for understanding the onset and treatment of these diseases. Lastly, we will identify the molecular defects that give rise to the loss of vacuolar acidity with repeated cell divisions, using a combination of cell biological, biochemical and genetic approaches. Given the link between the lysosome and a number of age-associated diseases, including Alzheimer's disease, the detailed level of knowledge about loss of vacuole acidification that we develop in this proposal, where the tools for dissecting this process are unparalleled, may provide new insights about the onset of these diseases. PUBLIC HEALTH RELEVANCE: The major non-infectious diseases that afflict humans are associated with cellular damage and organelle dysfunction. We have developed a system to study the molecular mechanisms that underlie these same changes in a simple organism, the yeast Saccharomyces cerevisiae. These studies have the potential to provide insight into and strategies to overcome diseases such as neuropathies, diabetes and muscle deterioration in humans.